This invention relates to methods for determining the presence of a target polynucleotide in a sample. In particular, this invention relates to methods for determining the presence of a target polynucleotide by real-time monitoring of an amplification reaction, preferably the polymerase chain reaction (PCR) using luminescent oxygen channeling immunoassay (LOCI) technology.
The sensitive detection of nucleic acids in a clinical sample opened a new era in the diagnosis of infectious diseases and other fields. Powerful nucleic acid amplification and detection methods are available which allow the detection of very small copy numbers of target polynucleotides. Tremendous progress has been made concerning the qualitative detection of nucleic acids, but the quantitative detection is still a challenge for the existing methods, especially for amplification methods based on the exponential amplification of a target polynucleotide. The best known amplification method of this type is the polymerase chain reaction (PCR). U.S. Pat No. 4,683,195; U.S. Pat No. 4,683,202.
Nucleic acids in a sample are usually first amplified by the amplification method and subsequently detected by the detection method. This sequential approach is based on a single end-point measurement after the amplification reaction is completed. The amount of amplified product observed at the end of the reaction is very sensitive to slight variations in reaction components because the amplification reaction is typically exponential. Therefore, the accuracy and precision of quantitative analysis using endpoint measurements is poor. Furthermore, endpoint measurements can produce a hook effect whereby high concentrations of a target polynucleotide to be amplified yield inaccurately low values.
In contrast to end-point determinations of amplified polynucleotides, real-time monitoring of amplification reaction product generation offers the possibility of better precision and accuracy in quantitative measurements because the measurements are taken during the exponential phase of the amplification process. In contrast to classical end-point measurements, multiple measurements are taken during real-time monitoring. During the exponential phase of the amplification process, none of the reaction components are limiting, and therefore the affect on accuracy of reaching a maximum signal are eliminated. Real-time monitoring of PCR is based on kinetic measurements offering a better and a more complete picture of the PCR process. A number of real-time monitoring methods have been developed, however the methods use fluorescent signals in all cases. This limits the earliest possible detection of amplifying DNA (RNA) because of the presence of unquenched or background fluorescence. The LOCI signal can be detected well before the fluorescent signal of, for instance, Roche""s TaqMan(copyright) fluorescent signal. See Heid et al., (1996) Genome Res., Vol. 6(10), pp. 986-994.
Nadau et al., U.S. Pat. No. 5,547,861 and Walker et al. U.S. Pat. Nos. 5,593,867 and 5,270,184, disclose a method for amplifying a target polynucleotide sequence called strand displacement amplification (xe2x80x9cSDAxe2x80x9d) and methods for detecting amplification products, including a real-time detection method using fluorescence polarization. In SDA temperature cycling is not required. Instead, the method relies on the ability of restriction enzymes to nick hemimodified DNA and relies on DNA polymerase to synthesize a complementary polynucleotide strand from the nick. Detection systems developed for SDA utilize displacement of a fluorescently-labeled detector probe DNA for real-time monitoring of the amplification reaction. A possible disadvantage of this system is that the probe is also a primer and any false priming (xe2x80x9cmisprimingxe2x80x9d) of this probe could lead to false positive signal generation. In contrast, the LOCI probes used herein are blocked at the 3xe2x80x2 terminal end and cannot be primers.
Numerous dyes have been developed for the detection of nucleic acids to detect a target polynucleotide after it has been amplified. For example, L. Lee et al., U.S. Pat. Nos. 5,863,727 and 5,800,996, describe fluorescent energy-transfer dyes, linkers for synthesizing these dyes, and methods that utilize the dyes. The patents describe the use of the dyes in nucleic acid reactions, including use of the dyes to detect the products of PCR reactions, after the end-point of a reaction and separation by electrophoresis. U.S. Pat. No. 5,863,727 col. 46 line 54. These patents do not disclose the used of the dyes in real-time monitoring of amplification products.
Chemiluminescent dyes, such as luminol and acridinium, and detection systems have been developed which offer the advantage of increased sensitivity over fluorescent systems. M. Lee et al., U.S. Pat No. 5,672,475 (""475 patent), disclose a method for performing end-point measurements of two substances, which theoretically could be polynucleotides, using two chemiluminescent conjugates. An essential feature of the invention of the ""475 patent is that each chemiluminescent molecule, eg. luminol and acridinium, is activated under a different set of conditions. The assay disclosed in the ""475 patent cannot be used to measure PCR reaction kinetics because the reactions require a separation step to remove unbound labeled conjugate, and therefore are not amenable to an all-in-one-tube assay. In addition, the assay method cannot be used in kinetic measurements of PCR reactions because the measurements require substantial changes in the reaction mixture to activate the chemiluminescent label, which are likely to affect the PCR reaction.
Law et al. U.S. Pat. Nos. 5,879,894, 5,395,752, and 5,702,887 (xe2x80x9cLaw patentsxe2x80x9d) describe test methods and long-emission wavelength chemiluminescent compounds for detecting two test substances in a single assay. U.S. Pat. No. 5,702,887 briefly mentions the use of two chemiluminescent compounds as labels in an amplification assay such as polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) (Col. 42, lines 31-57). However, although the disclosure indicates that the method could be used to quantitatively measure PCR products, it does not disclose the use of the method to make kinetic measurements of PCR reactions. In fact, the Law patents provide no examples of assays using the disclosed chemiluminescent compounds to measure polynucleotides. Luminescent oxygen channeling Immunoassays (LOCI) have been developed which offer the ability to measure large analytes, such as polynucleotides, with increased sensitivity in a homogeneous or heterogeneous format without the need of adding chemical reagents or heating the reaction to activate the luminescent compounds. U.S. Pat. No. 5,340,716, (Ullman, et al. 1994) (incorporated herein by reference). In LOCI a group which is bound to a specific binding pair member, such as a polynucleotide, is photochemically activated to a luminescent product and is used as a labeled reagent in assays for detection of an analyte, such as a target polynucleotide. The photochemical activation occurs by reaction with singlet oxygen that is generated by photochemical activation of a sensitizer. In the assay protocol the components are combined and the light produced after irradiation of the luminescent product is a function of analyte concentration.
The LOCI method was designed for the analysis of nucleotides in an end-point hybridization reaction. No study has suggested the use of LOCI in real-time monitoring of amplification reactions. It was possible that the kinetics of LOCI hybridization reactions, which involve hybridizations involving bead-coupled probes would be too slow to allow monitoring a PCR reaction. Moreover, the deleterious effect of singlet oxygen on DNA probes makes it problematic that LOCI utilizing a DNA probe would be effective after numerous, repeated illuminations. Finally, temperatures which allow formation of the LOCI complex on target DNA could have allowed unacceptable levels of mispriming, but this is not the case.
There remains a need for an assay method that utilizes an amplification reaction and that can be used for sensitive qualitative and quantitative measurements of a target polynucleotide. More specifically there remains a need for an assay method with the sensitivity of chemiluminescent detection of an amplification product, a wide dynamic range, and good precision and accuracy. To accomplish this, there remains a need for a detection method with rapid incubation and signal generation time to allow the real-time monitoring of an amplification reaction. Finally, there remains a need for an assay which can measure a target polynucleotide in an amplification reaction without a high-dose hook effect.
The current invention relates to the use of LOCI as the detection technology to monitor amplification reactions such as the polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), in an all-in-one-tube format. More specifically, the current invention involves the use of LOCI to measure the kinetics of a PCR reaction in an all-in-one assay format in order to quantitatively and qualitatively detect a target polynucleotide. The invention has the advantages of the sensitivity of chemiluminescence coupled with rapid signal generation to allow multiple measurements to be taken during the linear phase of a PCR reaction. This provides a more complete picture of the amplification process and sensitive qualitative and quantitative detection of nucleic acids with improved precision, accuracy, and a wider dynamic range.
The current invention relates to the use of luminescent oxygen channeling immunoassay (xe2x80x9cLOCIxe2x80x9d) technology to monitor amplification reactions, especially polymerase chain reactions (xe2x80x9cPCRxe2x80x9d). More specifically, the current invention involves the use of LOCI to measure the kinetics of a PCR reaction in an all-in-one assay format in order to quantitatively and qualitatively detect a target polynucleotide.
One embodiment of the invention is directed to a method for detecting the presence of a target polynucleotide in a sample comprising: (A) providing a reaction and detection mixture comprising in combination: (1) a sample; (2) a nucleic acid amplification system; and (3) a chemiluminescent detection system comprising a sensitizer capable of indirectly binding to the target polynucleotide and capable of generating singlet oxygen upon irradiation with light and a singlet-oxygen activatable chemiluminescent compound capable of indirectly binding to the amplified target nucleic acid; (B) amplifying said target polynucleotide through at least one amplification cycle; (C) allowing the indirect binding of said chemiluminescent compound and said sensitizer to said amplified target polynucleotide; (D) activating the sensitizer, wherein said activation of the sensitizer bound to the target polynucleotide causes the activation of said chemiluminescent compound bound to the target polynucleotide; and (E) determining the amount of luminescence generated by the activated chemiluminescent compound; (F) optionally repeating steps B-E; and (G) detecting the presence of said target polynucleotide by analyzing the amount of luminescence determined after at least one amplification cycle. Preferably, the sensitizer is a photosensitizer and the activation of the sensitizer comprises irradiation with light.
In another embodiment of the invention, the target polynucleotide comprises first and second complimentary strands; and the nucleic acid amplification system comprises: (1) a thermostable DNA polymerase; (2) 2xe2x80x2 deoxynucleoside-5xe2x80x2-triphosphates; (3) a forward-primer capable of binding to the first complimentary strand; and (4) a reverse-primer capable of binding to the second complimentary strand in a position that will direct DNA synthesis toward the site of annealing of the forward-priming oligonucleotide. The amplification system preferably utilizes the polymerase amplification reaction. If desired, thermal labile antibody against the thermal stable DNA polymerase may be used in a xe2x80x9chot startxe2x80x9d amplification reaction.
In yet another embodiment of the invention, the chemiluminescent detection system further comprises: (i) a first linking oligonucleotide capable of binding to both the target polynucleotide and a chemiluminescer-associated oligonucleotide; and (ii) a second linking oligonucleotide capable of binding to both the target polynucleotide and a sensitizer-associated oligonucleotide. If desired, guanosine residues may be replaced with inosine residues in one or both of the first and second linking probes. The sensitizer and sensitizer-associated oligonucleotide are associated with a first solid support while the singlet-oxygen activatable chemiluminescent compound and the chemiluminescer-associated oligonucleotide are associated with a second solid support. Preferably, the first and second solid support are beads and the acceptor beads comprise thioxene, anthracene, and rubrene.
In a further embodiment of the invention, a method for quantifying the amount of target polynucleotide in a sample is provided. The amount of luminescence is related to the amount of target polynucleotide in the sample. The luminescence determinations are made during an exponential phase of the amplification process and involve (a) determining a threshold cycle number at which the luminescence generated from amplification of the target polynucleotide in a sample reaches a fixed threshold value above a baseline value; and (b) calculating the quantity of the target polynucleotide in the sample by comparing the threshold cycle number determined for the target polynucleotide in a sample with the threshold cycle number determined for target polynucleotides of known amounts in standard solutions.
In yet another embodiment of the invention, a method is provided for detecting the presence of a target polynucleotide in a sample, the target polynucleotide comprising a first and a second complimentary strand. The method comprises (a) providing a reaction and detection mixture comprising in combination: (1) a sample, (2) a thermostable DNA polymerase, (3) 2xe2x80x2 deoxynucleoside-5xe2x80x2-triphosphates, (4) a forward-primer capable of binding to the first complimentary strand, (5) a reverse-primer capable of binding to the second complimentary strand in a position that will direct DNA synthesis toward the site of annealing of the forward-priming oligonucleotide, and (6) a chemiluminescent detection system comprising a photosensitizer capable of indirectly binding to the target polynucleotide and capable of generating singlet oxygen upon irradiation with light and a singlet-oxygen activatable chemiluminescent compound capable of indirectly binding to the amplified target nucleic acid; (b) denaturing said target polynucleotide for an initial denaturation period; (c) denaturing said target polynucleotide for a cycle denaturation period; (d) incubating the reaction and detection mixture to allow indirect binding of said chemiluminescent compound and said photosensitizer to said amplified target polynucleotide; (e) irradiating the photosensitizer with light, wherein said irradiation causes the activation of said chemiluminescent compound bound to the target polynucleotide by the sensitizer bound to the target polynucleotide; and (f) determining the amount of luminescence generated by the activated chemiluminescent compound; (g) annealing said forward priming and reverse priming oligonucleotides to the target polynucleotide; (h) synthesizing polynucleotide strands complementary to said first and second complementary strands of said target polynucleotide, said synthesis being catalyzed by the thermostable DNA polymerase; (i) optionally repeating steps Cxe2x80x94H; and (j) detecting the presence of said target polynucleotide by analyzing the amount of luminescence determined after at least one amplification cycle.
These and other embodiments of the invention will become apparent in light of the detailed description below.